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Tricyclic Antidepressants in Serum or Plasma for HPLC Analysis
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of serum or plasma add internal standard* and
2mL of 100mM phosphate buffer (pH 6.0)
Mix/vortex
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
1mL of 100mM acetic acid
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Tricyclic Antidepressants
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Dry Eluate
Evaporate to dryness at <40°C
Reconstitute
Reconstitute with 200µL of ethyl acetate/DI H
2
O (1:3)
Mix/vortex vigorously for 30 seconds
Analysis
Column temperature: 30°C
Mobile phase: A: 0.1% formic acid
B: ACN + 0.1% formic acid
Gradient: 30 to 50% B in 15 minutes
Flow rate: 1mL/minute
Detection: UV at 254nm
Recommended HPLC Column
Part Number
Hypersil GOLD 5µm, 150 x 4.6mm
25005-154630
124
Forensic
Applications